Auditory function is dependent on the formation of a functional cochlea, which includes the auditory sensory epithelium, referred to as the organ of Corti, and the associated spiral ganglion neurons that provide afferent neuronal innervation to the organ of Corti. The organ of Corti contains at least 6 different types of cells including mechanosensory hair cells and non-sensory supporting cells. Hair cells, supporting cells and spiral ganglion cells are all derived from a limited region of the otocyst, an embryonic structure that develops adjacent to the hindbrain. Other regions of the otocyst normally go on to develop as non-sensory structures within the inner ear. Existing data suggests that individual cells become specified to develop as either neuroblasts that will give rise to the afferent neurons of the cochlea or a population of prosensory cells that will then become subdivided into hair cells and supporting cells. While recent work has begun to identify some of the molecular signaling pathways that regulate these developmental events, our understanding is still fairly limited. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. First, we examined the role of the transcription factor Pou3f4 in development of spiral ganglion neurons. Mutations in Pou3F4 lead to deafness in mice and humans even though this gene is not expressed in either hair cells or ganglion cells. Rather, Pou3f4 is expressed in the the mesenchymal cells that surround ganglion cell peripheral axons as they extend towards the hair cells. In particular, spiral ganglion peripheral axons form fascicles referred to as inner radial bundles. In Pou3f4 mutants, inner radial bundles are significantly disrupted. As a result, many spiral ganglion neurons fail to reach the hair cells and so the number of synapses between hair cells and spiral ganglion neurons is significantly decreased. An analysis of candidate genes that might be regulated by Pou3f4 identified the axon guidance molecule EphA4 as a possible target. Analysis of EphA4 mutant mice indicated a similar defect in inner radial bundle formation and addition of EphA4 was sufficient to rescue the axon guidance defects observed in Pou3f4 mutants. Finally, chromatin immunoprecipitation demonstrated that Pou3f4 binds directly to the EphA4 promoter, indicating a direct role for Pou3f4 in the expression of EphA4. In a separate series of experiments we have examined the role of insulin-like growth factors (IGFs) in cochlear development. Previous work has demonstrated a role for Igfs in development of the spiral ganglion, but its role in the cochlea was unknown. To examine this, we generated mouse mutants lacking the primary IGF receptor, IGf1r. While these animals die at birth, we were able to analyze their cochleae at E18.5. Cochleae from Igf1r mutants were shorter than normal, had a reduced number of hair cells and defects in cellular patterning within the cochlea. To determine the bases for this defects, we used an in vitro system that allowed us to specifically examine changes in protein expression in response to inhibition of IGF signaling. Results indicated that many of the defects that occur when IGF signaling is inhibited are a result of a delay in the expression of the transcription factor Atoh1. Since Atoh1 is required for highly stereotyped development of hair cells, a delay in Atoh1 expression could lead to subsequent defects in cellular patterning. Moreover, subsequent experiments demonstrated that IGF signaling acts through the intracellular modulator, AKT, to influence cochlear development. Another project in the laboratory examined the role of Rbpj, a transcriptional regulator of Notch signaling, in the inner ear. Notch signaling is known to play myriad roles in inner ear formation. However, its role in the early specification of prosensory cells, the cells that will give rise to the hair cell sensory patches, was unclear. Deletion of the notch ligand Jagged1, causes a decrease in the size of the prosensory patches. But because other notch ligands are also expressed in the ear, the effects of completely eliminating notch signaling were not known. Rbpj is believed to be required for all notch signaling. Therefore, its deletion should lead to a complete loss of notch signaling. Deletion of Rbpj did lead to a nearly complete loss of all hair cell epithelia. To determine the role of notch in this pathway, the expression of several different markers of prosensory cells were examined. Results indicated that prosensory cell formation is normal in Rbpj mutants, but that without Rbpj prosensory cells fail to maintain expression of genes that are required for their formation and ultimately begin to die. In a project initially described last year, we demonstrated that three different transcription factors, Neurog1, Neurod1 and Sox2 are both required for development of the spiral ganglion and can induce non-sensory regions of the inner ear to develop as neurons. These cells express neuronal markers, develop morphologies that are consistent with neurons and can generate action potentials. Interestingly, the ability of these cells to develop as neurons is lost during the late embryonic period in mice. A key step in cochlear formation is the outgrowth of the cochlear duct. Moreover, this outgrowth also plays a role in the establishment of the precise pattern of hair cells and supporting cells within the organ of Corti. To begin to understand how this patterning occurs, we used genetic techniques to fluorescently label small numbers of developing hair cells or supporting cells. We then used time-lapse microscopy to observe these cells as they became patterned . Results indicate active movements in both cell types, but also that there are significantly different types of movements depending on the cell type. Hair cells undergo more directed movements while supporting cells seem to actively search their environment in all directions before moving in any particular direction. In collaboration with Richard Chadwick in the Section on Auditory Mechanics, we have examined changes in the mechanical properties of developing pillar cells and hair cells. As a mechanosensitive organ that is constantly being vibrated in response to sound, the structural mechanics of the organ of Corti must play an important role in auditory function. However, the factors that mediate cell stiffness within the cochlear are unknown. To address this, atomic force microscopy was combined with pharmacological manipulations of actin and microtubules to demonstrate that hair cell stiffness is dependent on actin while pillar cell stiffness is dependent on microtubules.